The present invention relates to novel glucoamylase variants (mutants) of parent AMG, in particular with improved thermal stability and/or increased specific activity suitable for, e.g., starch conversion, e.g., for producing glucose from starch. More specifically, the present invention relates to glucoamylase enzyme variants and the use of such variant enzymes.
Glucoamylase (1,4-xcex1-D-glucan glucohydrolase, EC 3.2.1.3) is an enzyme which catalyzes the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules. Glucoamylases are produced by several filamentous fungi and yeasts, with those from Aspergillus being commercially most important.
Commercially, the glucoamylase enzyme is used to convert corn starch which is already partially hydrolyzed by an xcex1-amylase to glucose. The glucose is further converted by glucose isomerase to a mixture composed almost equally of glucose and fructose. This mixture, or the mixture further enriched with fructose, is the commonly used high fructose corn syrup commercialized throughout the world. This syrup is the world""s largest tonnage product produced by an enzymatic process. The three enzymes involved in the conversion of starch to fructose are among the most important industrial enzymes produced.
One of the main problems that exist with regard to the commercial use of glucoamylase in the production of high fructose corn syrup is the relatively low thermal stability of glucoamylase. Glucoamylase is not as thermally stable as xcex1-amylase or glucose isomerase and it is most active and stable at lower pH""s than either xcex1-amylase or glucose isomerase. Accordingly, it must be used in a separate vessel at a lower temperature and pH.
Glucoamylase from Aspergillus niger has a catalytic (aa 1-xe2x80x2) and a starch binding domain (aa 509-616) separated by a long and highly O-glycosylated linker (Svensson et al. (1983), Carlsberg Res. Commun. 48, 529-544, 1983 and (1986), Eur. J. Biochem. 154, 497-502). The catalytic domain (aa 1-471) of glucoamylase from A. awamori var. X100 adopt an (xcex1/xcex1)6-fold in which six conserved xcex1xe2x86x92xcex1 loop segments connect the outer and inner barrels (Aleshin et al. (1992), J. Biol.Chem. 267, 19291-19298). Crystal structures of glucoamylase in complex with 1-deoxynojirimycin (Harris et al. (1993), Biochemistry, 32, 1618-1626) and the pseudotetrasaccharide inhibitors acarbose and D-gluco-dihydroacarbose (Aleshin et al. (1996), Biochemistry 35, 8319-8328) furthermore are compatible with glutamic acids 179 and 400 acting as general acid and base, respectively. The crucial role of these residues during catalysis have also been studied using protein engineering (Sierks et al. (1990), Protein Engng. 3, 193-198; Frandsen et al. (1994), Biochemistry, 33, 13808-13816). Glucoamylase-carbohydrate interactions at four glycosyl residue binding subsites, xe2x88x921, +1, +2, and +3 are highlighted in glucoamylase-complex structures (Aleshin et al. (1996), Biochemistry 35, 8319-8328) and residues important for binding and catalysis have been extensively investigated using site-directed mutants coupled with kinetic analysis (Sierks et al. (1989), Protein Engng. 2, 621-625; Sierks et al. (1990), Protein Engng. 3, 193-198; Berland et al. (1995), Biochemistry, 34, 10153-10161; Frandsen et al. (1995), Biochemistry, 34, 10162-10169.
Different substitutions in A. niger glucoamylase to enhance the thermal stability have been described: i) substitution of xcex1-helical glycines: G137A and G139A (Chen et al. (1996), Prot. Engng. 9, 499-505); ii) elimination of the fragile Asp-X peptide bonds, D257E and D293E/Q (Chen et al. (1995), Prot. Engng. 8, 575-582); prevention of deamidation in N182 (Chen et al. (1994), Biochem. J. 301, 275-281); iv) engineering of additional disuiphide bond, A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; and v) introduction of Pro residues in position A435 and S436 (Li et al. (1997), Protein Engng. 10, 1199-1204. Furthermore Clark Ford presented a paper on Oct. 17, 1997, ENZYME ENGINEERING 14, Beijing/China Oct. 12-17, 1997, Abstract number: Abstract book p. 0-61. The abstract suggests mutations in positions G137A, N20C/A27C, and S30P in a (not disclosed) Aspergillus awamori glucoamylase to improve the thermal stability.
Additional information concerning glucoamylase can be found on an Internet homepage
(http://www.public.iastate.edu/xcx9cpedro/glase/glase.html) xe2x80x9cGlucoamylase WWW pagexe2x80x9d (Last changed Aug. 10, 1997 ) by Pedro M. Coutinho discloses informations concerning glucoamylases, including glucoamylases derivable from Aspergillus strains.
Chemical and site-directed modifications in the Aspergillus niger glucoamylase are listed.
The object of the present invention is to provide improved glucoamylase variants with improved thermostablility and/or increased specific activity suitable for use in, e.g., the saccharification step in starch conversion processes.
The term xe2x80x9ca glucoamylase variant with improved thermostabilityxe2x80x9d means in the context of the present invention a glucoamylase variant which has a higher Txc2xd (half-time) than the corresponding parent glucoamylase. The determination of Txc2xd (Method I and Method II) is described below in the xe2x80x9cMaterials and Methodsxe2x80x9d section.
The term xe2x80x9ca glucoamylase variant with increased specific activityxe2x80x9d means in the context of the present invention a glucoamylase variant with increased specific activity towards the xcex1-1,4 linkages in the saccharide in question. The specific activity is determined as kcat or AGU/mg (measured as described below in the xe2x80x9cMaterials and Methodsxe2x80x9d section). An increased specific activity means that the kcat or AGU/mg values are higher when compared to the kcat or AGU/mg values, respectively, of the corresponding parent glucoamylase.
The inventors of the present invention have provided a number of improved variants of a parent glucoamylase with improved thermostability and/or increased specific activity in comparison to the parent corresponding enzyme. The improved thermal stability is obtained by substituting selected positions in a parent glucoamylase. This will be described in details below.
In the present description and claims, the conventional one-letter and three-letter codes for amino acid residues are used. For ease of reference, glucoamylase variants of the invention are described by use of the following nomenclature:
Original amino acid(s):position(s):substituted amino acid(s)
According to this nomenclature, for instance the substitution of alanine for asparagine in position 30 is shown as:
Ala30Asn or A30N
a deletion of alanine in the same position is shown as:
Ala30* or A30*
and insertion of an additional amino acid residue, such as lysine, is shown as:
Ala30AlaLys or A30AK
A deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or xcex94(A30-N33).
Where a specific glucoamylase contains a xe2x80x9cdeletionxe2x80x9d in comparison with other glucoamylases and an insertion is made in such a position this is indicated as:
*36Asp or *36D
for insertion of an aspartic acid in position 36
Multiple mutations are separated by plus signs, i.e.:
Ala30Asp+Glu34Ser or A30N+E34S
representing mutations in positions 30 and 34 substituting alanine and glutamic acid for asparagine and serine, respectively. Multiple mutation may also be separated as follows, i.e., meaning the same as the plus sign:
Ala30Asp/Glu34Ser or A30N/E34S
When one or more alternative amino acid residues may be inserted in a given position it is indicated as
A30N,E or A30N/E, or A30N or A30E
Furthermore, when a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Thus, for instance, when a modification of an alanine in position 30 is mentioned, but not specified, it is to be understood that the alanine may be deleted or substituted for any other amino acid, i.e., any one of:
R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.